Ribozymes are RNA molecules that possess RNA catalytic ability (see Cech, 1986, 1990, for review) that cleave a specific site in a target RNA substrate in a trans reaction. The catalytic ability is shown in that the number of RNA molecules that are cleaved by a ribozyme is greater than the number predicted by stochiochemistry (Hampel and Tritz, 1989; Uhlenbeck, 1987).
Ribozymes catalyze the phosphodiester bond cleavage of RNA and are emerging as a new tool for controlling the cellular RNA levels of specific genes. Several ribozyme structural families have been identified including Group I introns, RNase P, the hepatitis delta virus ribozyme, hammerhead ribozymes and the hairpin ribozyme originally derived from the negative strand of the tobacco ringspot virus satellite RNA (sTRSV) (Sullivan, 1994; U.S. Pat. No. 5,225,347, columns 4-5). The latter two families are derived from viroids and virusoids, in which the ribozyme is believed to separate monomers from oligomers created during rolling circle replication (Symons, 1989 and 1992). Hammerhead and hairpin ribozyme motifs are most commonly adapted for trans-cleavage of mRNAs or viral RNA genomes for gene therapy (Sullivan, 1994).
U.S. Pat. No. 5,093,246 issued to Cech et al. discloses an endoribonuclease that has an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place, with a requirement for free guanosine or guanosine derivatives. The limited number of nucleotides available for hybridization to an RNA substrate has been found to limit the efficiency of the Cech endoribonuclease. A number of nucleotides in the active site of the Cech endoribonuclease have been found to need to be conserved for efficient endoribonuclease activity. This restricts the number of permutations of active site sequences which can be engineered to effect hybridization to target sequences, thereby restricting the range of RNA target sequences clearable by the Cech endoribonuclease. The Cech endoribonuclease also modifies the RNA substrate by adding a free guanosine nucleotides to the 5' end of cleaved RNA.
U.S. Pat. No. 5,254,678 issued to Hasseloff et al. discloses a hammerhead ribozyme. The catalytic region in the hammerhead ribozyme is in reverse order to that of the hairpin ribozyme as described herein below. The hammerhead ribozyme is not as efficient in vivo as the hairpin ribozyme. The hammerhead ribozyme has not been approved for human clinical trials.
A hairpin ribozyme has been developed by the applicants which successfully cleaves HIV-1 genomic and mRNAs in vitro and in cells (Ojwang et al., 1992; Yu et al., 1993). This HIV-1 specific hairpin ribozyme is being prepared for clinical testing in human AIDS patients (Wong-Staal, 1994). Given the therapeutic potential of the hairpin ribozyme, as well as the general importance of a designed "molecular knife", it would be useful to have additional hairpin ribozymes.
However, currently such ribozymes are identified empirically. It would be useful to have a method whereby any nucleotide sequence can be screened to identify new sequences with catalytic activity. Further, hairpin ribozymes that are currently available cleave an RNA substrate within the catalytic region at a site which has a GUC sequence following the site of cleavage. It would be useful to have hairpin ribozymes which can cleave at additional sites.